Biology of the NMDA Receptor.

Chapter 5Transcriptional Regulation of NMDA Receptor Expression

Guang Bai and Peter W. Hoffman.

5.1. INTRODUCTION

The N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors are intimately involved in a number of important neuronal activities in mammalian nervous systems including neuronal migration, synaptogenesis, neuronal plasticity, neuronal survival, and excitotoxicity. Through these activities, NMDA receptors (NRs) play an important role in the development of drug addiction, pain perception, and the pathogenesis of neurological disorders such as schizophrenia and Huntington’s disease [1–10].

It is generally believed that aberrant or pathological NR effects occur mainly via abnormal receptor activity, resulting from altered availability of agonists or modified quality or quantity of membrane-associated receptors. In mammals, functional NRs are heterotetramers of subunits encoded by three gene families, i.e., NMDAR1 (NR1 or Grin1), NMDAR2 (NR2 or Grin2), and NMDAR3 (NR3 or Grin3) [3,4,11]. The NR1 family has one gene; the NR2 family has four (designated A through D); and the NR3 family has two (A and B). Structurally, NR1 is an essential component found in all tetramers, while different NR2 members are incorporated based on age and nervous system region. NR3 proteins function as negative components when included in the structures [3,4,11,12]. Eight variants of NR1 protein are produced by alternative splicing and distributed differentially in nervous systems [13–15]. This complex composition of different subunits and splicing variants forms the primary basis of the functional diversity of NRs.

From January 1992 to June 2007, more than 1000 research articles relevant to NR expression were published. In sum, they concluded that the expression of NR genes is cell- or tissue-specific, relatively stable, and regulated differentially by various physiological, pharmacological, and pathological factors. Most of these conclusions were based on assessments of changes of the steady state levels of mRNA and protein that may be driven by numerous sophisticated mechanisms. Transcription is the initial step and generally the most sensitive to cellular needs and environmental cues. Thus, it serves as a major mechanism controlling gene expression [16].

Precise spatial and temporal expression of a selective set of genes determines phenotypic differences among distinct tissues and cells in higher eukaryotes [16–18]. In the case of the NR gene families, transcription of each subunit gene in a given neuron or cell must be coordinately controlled but differentially responsive to cell type, developmental stage, and environmental signals to maintain healthy cellular function. How this coordinated control takes place is an important and challenging question. This chapter reviews studies that explore the transcriptional control of NR genes. It discusses studies of promoter and regulatory sequences, regulatory units, developmental regulation, cell type specificity, growth factor regulation, neurological disorders, and epigenetic mechanisms.

5.2. IDENTIFICATION OF PROMOTER

The promoter is the gene component that directs transcription [17,19]. It consists of a core or basal promoter that spans the most upstream and nearby transcription start sites (TSSs) to initiate transcription as well as regulatory regions containing enhancers or silencers to regulate transcription rate [17,20]. It had been assumed that tissue-specific and/or -inducible genes in higher eukaryotes contained a single TSS and an immediate upstream TATA or CAAT box. Multiple TSSs, GC-rich regions, and structures lacking TATA and CAAT were viewed as characteristics of housekeeping genes [18,21–23]. Another early concept was that most eukaryotic genes utilized a single promoter and that the core promoter contained several basic DNA binding sequences [16,22,24,25]. This concept has been modified significantly [16,20], particularly since large-scale analysis of transcription regulatory regions became possible [19,26–28].

As shown in Figure 5.1, more than 50% of human genes including many neuronal genes, utilize multiple TSSs from a single exon or from different exons. In the latter case, multiple promoters control a single gene (>20% of human genes) [28]. A significant number of promoters start from the exonic sequence of another gene [19]. Many genes exert promoter activities at their 3′ ends, possibly for antisense transcription or for transcription of a downstream gene [29]. Bidirectional promoters exist for many genes (~11% in humans) [30]. Supported by additional evidence [19,26–29,31], GC-rich structures and those lacking TATA and CAAT boxes are associated with many tissue-specific genes including neuronal genes. Based on these observations, our view of promoter structure and function has been simplistic.

Current view of transcription and gene expression. The flow of gene expression from the nucleus to the cytoplasm is shown. Emerging concepts of transcription and gene expression are indicated by circled numbers. (1) Multiple TSSs for more than 50% genes, (more...)

Predication of promoters is difficult because no universal consensus may be employed [16]. Despite this difficulty, computer algorithms have been developed and most are available online [32–36]. Identification of bona fide promoters still relies largely on experimental mapping TSSs or the 5′ ends of mRNA [16]. Several databases have been established to collect promoters identified in experiments [37–39]. Databases of full-length cDNAs are also available although many of the included sequences have artificial or incomplete 5′ ends [40–42].

5.2.1. Mapping Transcription Start Sites

The 5′ end of a given mRNA is also the TSS on genomic DNA. In experiments, the TSS is usually defined by mapping the 5′ end of the mRNA followed by an alignment to the genomic sequence. Conventional methods include RNase protection assays, primer extension, cell-free in vitro transcription, and 5′-rapid amplification of cDNA ends coupled to sequencing of cloned ends. Several novel methods have been developed for a large-scale analysis of the 5′ ends, e.g., 5′ end serial analysis of gene expression [43] and cap analysis of gene expression [44].

NR1 was the first glutamate receptor gene to be mapped for its 5′ end [45]. Two major clusters of TSSs separated by 40 base pairs (bps) were identified from a GC-rich and TATA and CAAT boxless region. Similar conclusions were reached by later studies of NR1 gene from humans [46] and chicks [47]. Rat NR2A TSSs were identified following systematic RNA mapping [48]. Multiple TSSs were found to spread almost entirely across exon 1. Similar results were obtained for mice [49] and humans [50]. Only one TSS was found for the NR2B gene in mice [51,52] and humans [53]. Two TSSs separated by 18 bps were mapped for the mouse NR2C gene [54], and the downstream TSS was more highly utilized. All mapped TSSs of NR genes are located within a single exon. No current reports focus on mapping of the NR2D and NR3A/B TSSs.

Recent genome-wide analysis has shown that TSS selection is tissue-dependent for many genes [55]. The proximal TSS cluster of the NR1 gene is mostly recognized in the brain, while the distal TSS cluster is heavily utilized by PC12 cells that express NR1 mRNA but not detectable NR1 protein [56]. Therefore, it has been proposed that the additional 5′ untranslated region (5′ UTR) sequences transcribed from the distal TSSs interfere with translation initiation [57]. However, whether the transcription from the distal site is part of a bona fide control mechanism or simply an aberration of the PC12 cell line is still unclear.

5.2.2. Functional Analysis

All NR genes mapped for TSSs have been functionally tested for minimal sequences that govern transcription initiation. The most sensitive and convenient means to test promoter function is reporter gene technology that allows a putative promoter to drive expression of an easily assayable foreign gene in cultured cells or in transgenic animals [58].

For the rat NR1 gene, the basal or core promoter has been defined within the −1 to −356 bp fragment by luciferase reporter gene experiments in cultured cell lines and primary neurons [59,60]. The addition of upstream sequence up to −5.4 kb significantly increased promoter activity, suggesting that this region contains enhancer elements [61].

Activity of the NR2A promoter has been tested in cultured cells and primary neurons in rats [48], in transgenic mice [49], and in cultured human neuroblastoma cells [50]. Interestingly, the rat NR2A core promoter elements were restricted within exon 1 (1140 bps). These sequences alone demonstrated cell type selectivity with much stronger activity in neurons than in glial or HEK293 cells [48]. In addition, DNA sequences between the upstream and downstream TSSs retain comparable or even stronger ability to drive luciferase expression in comparison to the upstream genomic sequences [48]. These sequences may represent a novel type of multiple promoter, but the issue remains unexplored. In transgenic mice, an equivalent sequence from the mouse NR2A gene (~1 kb) directed luciferase expression selectively in the brain with activity comparable to a fragment extending −9 kb upstream [49].

The minimal promoter for the NR2B gene was found within a short fragment of −106 to +158 in NIH3T3 cells but its cell type specificity requires further analysis [51]. However, a larger fragment up to −572 bp of this gene showed neuron-specific activity in the brains of transgenic mice [52]. The NR2C core promoter has been defined within a short sequence (−64 to +203 relative to the most upstream TSS) in cultured cells [62]. No promoter activity has been examined for the NR2D and NR3A/B genes.

5.3. REGULATORY UNITS: CIS ELEMENTS AND TRANS FACTORS

Nuclear proteins regulate transcription by binding to specific sequences in the regulatory region of the target gene. These sequences were initially identified by their interactions with trans factors. Analysis of these sequences indicated that cis elements share conserved consensus motifs (typically, 6 to 10 bps) [16,18]. To date, five types of cis elements have been proposed: enhancers, silencers, insulator/boundary elements, and locus control regions [63].

Transcription factors interact with sequences in enhancers or silencers to turn transcription on and off or manipulate the efficiency of the cognate promoter. Most of these binding sites are located within the 5′ upstream region in clusters to form enhancers that positively impact transcription or repressors that negatively regulate the promoters. However, some cis elements can be found in other regions of the gene or genome [16,17].

Studies in vitro and in living cells demonstrated that one type of cis element may interact with different groups of trans factors, and conversely one type of trans factor may bind different types of cis elements [16]. We shall summarize the interactions of trans factors and cis elements in NR genes, considering each relevant transcription factor or family individually. Figure 5.2 presents the relative positions of the analyzed binding elements on the relevant genes for humans, rats, and mice. Although a number of putative cis elements have been proposed to these promoters on the basis of motif searches, their functionality has not been demonstrated and will not be discussed here.

5.3.1. Specific Protein (Sp) Family

Specific protein 1 (Sp1) is the prototype of the Sp family that includes eight additional members (Sp2 to Sp9) [64–67]. Sp proteins bind GC (5′-GGGGCGGGG) or GT/CACC (5′-GGTGTGGGG) boxes. Sp1 is expressed during neuronal differentiation [61] and deletion of this factor from the genome results in aberrant brain development in the embryo and eventual lethality [68].

A motif search of functional promoter sequences revealed putative GC boxes near TSSs of the NR1, NR2A, NR2B, and NR2C genes. Two tandem GC boxes immediately upstream of the TSSs of the NR1 gene have been analyzed functionally by a series of sophisticated experiments [56,59]. These elements were shown to be responsible for growth factor upregulation of the promoter [56]. Factors interacting with this sequence include Sp1, Sp3, Egr1, and MAZ [59,69]. Surprisingly, Sp1 and Sp3 also bind an NFκB site 3 kb upstream of the TSSs in the NR1 gene [70].

Three GC boxes from the core promoter of the NR2A gene were tested by electrophoretic mobility shift assay and reporter gene assay and shown to possess positive regulatory activity [48]. At least three GC boxes were identified from the NR2B promoter. Although all three interacted with nuclear proteins, they may be redundant in upregulating the promoter since a fragment bearing only one such site showed promoter activity comparable to one having all three [51]. One GC-rich sequence harboring two tandem GC boxes was found immediately upstream of the NR2C TSS. This sequence binds Sp proteins and positively regulates the reporter gene [62].

Sp factor, specifically Sp1, often collaborates with other transcription factors by direct interaction to enhance or reduce its active role on the promoter. On the NR1 promoter, a brain-specific factor known as MEF2C was found to interact directly with Sp1 and synergize promoter activity [60]. Interestingly, this interaction is independent of MEF2C binding to the promoter since forced expression of MEF2C with a promoter lacking the MEF2 site retains a similar impact on the promoter as long as Sp1 factor is coexpressed.

5.3.2. MAZ

Myc-associated zinc finger protein (MAZ) binds the same cis element as Sp1 [71]. In the NR1 promoter, this protein was shown to compete with Sp1 at the proximal GC boxes [69]. Since GC boxes are common cis elements in the regulatory regions of genes in the nervous system, the interactions of transcription factors from different families with this element suggest that an appropriate balance in the interactions of various trans factors and these sites is important to maintain neuronal function [53,72,73].

5.3.3. Early Growth Response (Egr) Family

Early growth response (Egr) proteins belong to the immediate early gene family of transcription factors and are encoded by four genes, i.e., Zif268/Egr1/Krox-24, Erg2, Egr3, and Egr4 [74,76]. These proteins are inducible and transiently expressed in most tissues including neurons in response to environmental cues such as neurotrophins [74] and glutamate [75]. Egr proteins recognize an Egr response element (GSG, 5′-GCG5CG-3′) proximal to the TSSs of target genes and usually function as activators.

The rat NR1 promoter has a perfect GSG site immediately upstream of the TSSs [45]. Motif searches of genome databases revealed that human and mouse NR1 genes also bear this motif in similar locations. This site responds to growth factor stimulation in PC12 cells, binds recombinant Egr1 and Egr3, and enhances reporter gene expression in response to coexpressed Egr1 and Egr3 [74]. Therefore, the GSG site is believed to mediate at least part of the positive effect of nerve growth factor (NGF) on NR1 expression [56].

5.3.4. Jun and Fos Families

Jun and Fos are among the most studied transcription factors [77]. Their expression is associated with many cellular activities such as growth, differentiation, stress, and apoptosis [1,77]. Members of the Fos, Jun, and ATF subfamilies form various homodimers or heterodimers and interact with the activator protein 1 (Ap1) consensus site of 5′-TGA(C/G)TCA to regulate transcription rate. Dimer composition is context-dependent and may significantly influence activity [78]. An active Ap1 site in the distal promoter of the NR1 gene was initially identified by computer alignment and subsequently confirmed by DNA–protein binding assays and reporter gene experiments [45]. An Ap1 site in the NR2B promoter has been suggested to mediate positive effects of ethanol-induced expression of this gene [79]. This Ap1 site is also recognized by factors in the CREB family. Supershift EMSA experiments demonstrated that phosphorylated CREB is increased in DNA–protein complexes bound to the NR2B Ap1 site after ethanol treatment [79].

5.3.5. MEF2C

MEF2 is a subfamily of the MADS (MCM1 agamous deficiens serum response factor) family of DNA binding proteins consisting of four members (MEF2A, B, C, and D) that bind to the consensus of 5′-YTAW4TAR [80]. MEF2C is highly expressed in the developing brain in parallel with NR expression [81]. A MEF2 site (5′-TTATTTATAG) has been identified approximately 500 bps upstream of the GSG and GC-boxes in the NR1 promoter [60]. This site positively regulates the NR1 promoter in cultured cell lines, primary neurons, and differentiating neurons [60]. MEF2C is the major trans factor responsible for this upregulation. Surprisingly, it synergizes this effect with the Sp1 factor via the proximal tandem GC boxes. Considering the expression of MEF2C and Sp1 in developing brains and differentiating neurons [61], the interaction of these factors may be an important force driving expression of the NR1 gene during development.

5.3.6. CREB

In the nervous system, the cAMP response element (CRE) binding (CREB) protein receives signals primarily from the protein kinase A (PKA) pathway and from other pathways including the Ras-mitogen-activated protein kinase (MAPK) pathway triggered by NR activation [82]. Phosphorylated CREB proteins bind the highly conserved 8-bp palindromic CRE consensus (5′-TKACGTCA), and Ap1 or Ap2 sites [83]. CREs retaining the 3′ half of the palindromic consensus, while still active, are considered atypical and demonstrate less activity than full-size sites [84,85]. CREB has a large number of target genes and a database has been established to collect experimentally proven targets and predict potential CRE sites (http://natural.salk.edu/creb) [86].

The NR1 gene contains several atypical CRE sites around the TSSs and in the distal region [45,87]. In cultured embryonic neural cells, activation of the PKA pathway by forskolin increases both NR1 mRNA and protein. Chromatin immunoprecipitation (CHIP) experiments employing CREB antibody precipitated genomic DNA fragments proximal to the NR1 TSSs [87]. The CRE/CREB signal is often associated with neural activities such as synaptic plasticity [88] and nociception [89] in the mature central nervous system (CNS). The significance of these interactions in developing neurons remains to be explored.

In the NR2B regulatory region, a functional CRE site was found to be responsive to ethanol in cultured cortical neurons [90]. However, the changes observed for mRNA level in the treated neurons and for reporter gene activity in transfected cultures were marginal in comparison to the significant change in DNA binding of the CRE site by nuclear extracts following ethanol treatment.

5.3.7. NFκB Family

The NFκB family is composed of five transcription factors (p50, p52, p65, c-Rel, Rel-B) and associated with mechanisms of neuronal survival, neuronal plasticity, and neuropathology [91–93]. Homodimers and heterodimers of these proteins typically function as activators by binding to the consensus of 5′-GGGRDTYYCC. Analysis of the upstream sequence of the rat NR1 promoter revealed a perfect NFκB site. Unexpectedly, this site did not bind functional NFκB factors found in nuclear extracts of differentiating neurons [70]. More surprisingly, this NFκB site formed complexes with these nuclear extracts independent of any NFκB factor. Sp factors are the protein components of these complexes. The binding of Sp factors to the NR1 NFκB site was further confirmed in living cells by CHIP [70]. The binding strengths of different Sp factors to this site in living cells vary with neuronal differentiation. This kind of Sp factor binding to an NFκB site is the first such example in neuronal gene regulation. Considering the presence of the NFκB sites in a wide spectrum of neuronal genes and the universal expression of Sp factors in the brain, this finding may have broad implications in neuronal gene expression.

5.3.8. Tbr1

Tbr1 is a neuron-specific T box transcription factor expressed during brain development [94]. It binds a palindromic DNA consensus (5′-TSACACCTAGGTGTGAAATT) as well as nonpalindromic sequences homologous to either half side of the consensus such as 5′-YTTCACACCT [95]. NR1 and NR2B promoters both contain nonpalindromic T box elements. A combination of luciferase reporter assays and knock-out mice demonstrated a positive effect of Tbr1 on NR2B and/or NR1 expression [96]. Considering its expression in developing brain, Tbr1 is very likely an activator for the NR gene expression during development.

5.3.9. Estrogen Receptor

Estrogen regulates gene expression by interacting with its nuclear receptors, ERα and ERβ, or by activating signal transduction pathways through unidentified membrane- associated receptor(s) [97]. Ligand-bound homodimers of ERα and ERβ recognize an estrogen response element or ERE (5′-GGTCANNNTGACC) and usually upregulate the promoter. ERα or ERβ monomer binding of half ERE sides has been suggested but is still controversial [98]. Increasing evidence suggests that estrogen steroids play roles in several CNS functions such as synaptic plasticity and neuroprotection involving NR activity [97]. Ovarian steroid withdrawal by ovariectomy in rats produced NR hypoactivity, specifically in the hippocampus [99]. Estradiol treatment of the rats recovered hippocampal NR ligand binding preceded by changes in NR1 and NR2B mRNA levels, visualized by in situ hybridization, suggesting that estrogen may regulate NR gene transcription.

Although the effects of estrogen on the NR1 and NR2B promoters have not been tested directly, the NR1 promoter is upregulated by the Ras-MAPK pathway that can be activated by estrogen [97,100,101]. In addition, expression of ERα and ERβ in the brains of female mice was well correlated with NR2D mRNA levels by Watanabe et al. [102] These authors identified four half palindromic ERE (5′-TGACC) sites in the 3′ UTR of NR2D mRNA. The capability of these half sites to regulate transcription was further tested using a hetero promoter–reporter gene assay in cultured cells [103]. However, the TGACC half site sequence is very common in the genome and therefore the bona fide NR2D promoter must be tested to confirm the estrogen effect.

5.3.10. REST/NRSF

An effort to delineate the mechanism underlying neuronal transcription revealed a group of cis elements (consensus: 5′ DYCAGCACCNNGGACAGNNNC) from the regulatory regions of many neuronal genes—designated repressor element 1 (RE1) or neuron-restrictive silencer element (NRSE) [104–108]. The cognate trans factor is RE1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF), a zinc finger protein with several short isoforms generated by alternative splicing [106]. REST expression is high in nonneuronal cells and neural progenitors where it acts with cofactors to suppress neuronal genes [106,109–111].

Putative RE1/NRSE sites have been found bioinformatically and experimentally in the regulatory regions of the NR1, NR2A, and NR2B genes [52,104,106,107]. A global search of REST targets by ChIPSeq uncovered REST bound sequences near the NR2A, 2C, and 3A genes in a human T lymphoblast cell line [108]. Another genome-wide analysis coupling a large-scale CHIP with serial analysis of chromatin occupancy revealed that REST occupied one or more sites linked to each of the NR1, NR2C, and NR2D genes in a cultured mouse kidney cell line [112].

The functional impacts of REST and identified RE1 sites on bona fide promoters in a reporter gene assay have been studied only for the NR1 and NR2B genes [61,113,114]. The NR1 RE1 element was shown to bind REST and negatively regulate the promoter in nonneuronal and neuroprogenitor cells [61]. The RE1 site in the 5′ flanking region of the NR2B gene also demonstrated a negative regulatory effect on the promoters in embryonic neural cells [114].

5.4. DEVELOPMENTAL EXPRESSION OF NR GENES

Development is the cellular program that produces differentiated tissues and organs from undifferentiated precursors. It has been proposed that a network of interactions between cis elements in DNA and grouped trans factors expressed following specific temporal and spatial patterns is a key driving force for this program [115–117].

NR genes exhibit heterogeneous expression in the developing brain. The mRNAs of NR1, NR2A, and 2D become detectable in CNS following embryonic day 14 in rodents [118,119]. During the first 3 postnatal weeks, NR1 is progressively upregulated in the whole brain, followed by NR2A expression. In contrast, NR2B shows high expression in the whole brain within the first 2 postnatal weeks. Subsequently, it decreases in the cerebellum, but remains highly expressed in the forebrain. NR2C is expressed at low levels in the early stages of development and shows progressively higher expression in the cerebellum and olfactory bulb following postnatal day 11. NR2D is highly expressed during the first postnatal week throughout the brain, then declines and becomes restricted mainly to the middle brain in adults [118–120]. NR3A mRNA appears in the rat CNS by E16, becomes robustly expressed in the whole CNS except the forebrain by E19, and peaks by P7 in rats. Its expression then declines significantly, and is limited to a few CNS areas such as the spinal cord and thalamus [121,122]. NR3B expression emerges in motor neurons of the spinal cord by P10 and reaches its maximum by P21 [123].

The impact of cis elements on developmental expression of the NR1 promoter has been analyzed in more detail than impacts on other NR genes [61,70]. In P19 embryonic stem cells, changes in NR1 mRNA level and promoter activity are coordinated with neurogenesis and neuronal differentiation. Additionally, REST expression and binding to the NR1 RE1 site are robustly downregulated in the early stages of neurogenesis and differentiation [61]. A 2-day gap between REST downregulation and upregulation of the NR1 gene promoter suggests that other factors are involved. Experiments with cis element mutations suggest that the GC box/GSG, MEF2 and NFκB sites and relevant trans factors are important for promoter activation following this 2-day gap although the mechanism underlying the gap is unknown [60,70].

NR2A and 2B promoter fragments have been studied in transgenic mice to determine their total activity in the developing brain. The NR2B promoter up to −9 kb exhibited a 100-fold greater activity in neuronal and glial cocultures than in pure glial cultures. A sequence between −1253 and −1180 is necessary for the developmental expression of NR2A [49]. The activity of these elements during development has not been precisely studied [49,52].

To follow NR2C expression, Karavanova et al. recently developed a knock-in mouse model by inserting the bacterial LacZ gene into the 5′ end of the coding sequence of the endogenous NR2C gene [124]. They were able to map the regional and developmental expression of NR2C. Unfortunately, this model cannot provide details of how the promoter contributes to this expression. In a separate study, an NR2C promoter–LacZ fusion gene was integrated into the mouse genome and reporter expression was found in layer 4 spiny stellate cells of the adult barrel cortex [125] but no developmental information is available.

The mechanism underlying the decline of the NR2D gene during development remains unexplored. Recent data generated by genome-wide searches for REST targets linked DNA sequences bound by REST to the NR2C and NR2D genes [108,112]. Since the REST–RE1 interaction plays an important role in the developmental expression of neuronal genes, whether these identified RE1 sites participate in developmental expression and restriction of these two genes should be investigated.

5.5. GROWTH FACTOR REGULATION OF NR GENES

Growth factors are important extracellular stimuli that initiate and maintain neuronal differentiation and survival. Brain-derived growth factor treatment of cultured embryonic cortical neurons increased the NR1 mRNA level about two-fold [126]. Studies with PC12 cells showed that activity of the NR1 promoter is upregulated by several growth factors including NGF, fibroblast growth factor, and epidermal growth factor [56,60]. A recent report scrutinized several lines of PC12 cells and concluded that NGF upregulates NR1 mRNA in a cell line-dependent manner [127].

Based on these studies, NR1 transcription is likely upregulated by growth factors during neuronal differentiation. Further studies demonstrate that NGF utilizes both MAPK and phosphatidylinositol 3-kinase (PI3K) pathways to regulate the NR1 promoter [101]. Interestingly, MAPK targets included Sp1 and a group of unknown nuclear proteins specifically interacting with single-stranded DNA of the proximal region of the NR1 promoter [101,113].

Using proteomics, DNA binding, and siRNA technologies, we found that the major components of these complexes are hnRNPs that regulate mRNA transportation, splicing, and gene transcription. The downstream mediators of the PI3K effect have not yet been elucidated [101]. NR activation in the nervous system controls the transcriptional regulation of many genes such as those encoding growth factors through the Ras-MAPK pathway [128,129]. This reciprocal regulation of growth factor and NR genes may be part of the mechanism underlying the neurotrophic effect of NR activation.

5.6. CELL TYPE SPECIFICITY OF NR GENE EXPRESSION

Neurons are the major sites of NR expression. It is surprising that NR expression and receptor activities are also found in nonneuronal cells and peripheral tissues including glial cells [130], pancreatic islands [131], lungs [132], bone cells [131], adrenal medulla and kidneys [133], keratinocytes [134], and heart [135], although their exact functions in these tissues are not yet fully understood.

The neuronal specificity of NR1 expression has been shown to reside in the proximal promoter. Functional analysis disclosed that 356 bps of the NR1 core promoter confer high activity in neuronal PC12 cells and in cultured neurons, low activity in the C6 glioma, and almost no activity in HeLa cells [45,56,59]. As noted, this 356 bp proximal promoter contains a consensus RE1 site that negatively regulates this promoter in nonneuronal and neuroprogenitor cells [60,87,113]. A number of universal cis elements from proximal and distal regulatory regions have been found to positively regulate the NR1 promoter. The coordination of these positive and negative elements determines NR1 expression in different group of neurons.

The neuronal specificity of the NR2A gene is restricted by a fragment consisting of exon 1 and a few upstream sequences in transgenic mice. The addition of the upstream sequence up to 9 kb did not show additional effects [49]. The same observation has been repeated in cultured cells [48]. The core promoters of the NR2B and NR2C genes also exhibit neuronal specificity in transgenic mice and cultured neurons [49,52,62]. Since the RE1 sites identified for the NR2 genes in several studies are distal to these core promoters [106–108,112,114]. and no direct evidence indicates how these sites regulate bona fide promoters, whether RE1/REST is the major player of neuronal specificity of NR2 gene transcription must be reconsidered.

While NR proteins are expressed in several nonneuronal cell types, the mechanisms controlling this expression have not been directly investigated. Only tangential evidence exists. One study employing C6 glioma cells as negative controls found weak NR1 promoter activity in these cells. Nuclear extracts from C6 glioma cells were also found to bind the Sp1 sites of the NR1 promoter [59]. The control of NR expression in nonneuronal cell types deserves further scrutiny and would certainly provide interesting data on cell-specific transcriptional control and developmental pathways.

5.7. NEUROLOGICAL DISORDERS

NR-mediated glutamate toxicity is considered a common pathological pathway of many acute and chronic neurological disorders such as brain trauma, brain ischemia, schizophrenia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, AIDS dementia, and Lou Gehrig’s disease [6–8,10,136]. Since NR function and activity are determined by a specific set of NR genes expressed in a given cell and by the level of that expression, abnormal transcription of the NR genes may contribute to NR pathological effects.

5.7.1. REST, Huntingtin, and Huntington’s Disease

Alteration of NR expression may be one mechanism underlying the pathology of Huntington’s disease [137]. NR ligand binding is significantly decreased in the caudate in Huntington’s disease [138]. Wild-type huntingtin has been shown to retain REST protein in the cytoplasm, preventing it from silencing neuronal genes [139]. A mutated form of huntingtin genetically associated with Huntington’s disease [137] does not retain this function and releases REST that can suppress neuronal genes in mature neurons [139].

In situ hybridization of a Huntington’s disease brain revealed decreases in the NR1 and NR2B mRNAs correlated with severity [140]. Decreases of NR2A and NR2B mRNAs in the hippocampus were also observed in mouse models of Huntington’s disease (R6/2) [141]. NR2D is upregulated in neuronal nitric oxide synthase-positive interneurons in the caudates of these mice [142]. It is therefore likely that transcription of NR genes is suppressed by free REST in some neuronal nuclei of Huntington’s patients, while other cells may express inappropriate subunits.

5.7.2. Promoter Polymorphism and Schizophrenia

NR hypofunction has been reported in schizophrenic patients [143] and schizophrenic behaviors have been noted in NR2A-depleted mice [144]. Reduced expression of NR genes including the NR1, NR2B, and NR2C genes in the thalami of schizophrenic patients has also been documented [145,146].

The possibility that lowered promoter activity due to polymorphisms may contribute to NR hypofunction has been investigated [147–149]. In a case-controlled study (375 schizophrenics, 378 controls, equally divided by sex), the repeat length of a variable GT repeat in the NR2A promoter was correlated with mRNA reduction and the severity of chronic schizophrenia [50]. This initial survey was confirmed in an extended study with twice as many schizophrenics and controls [147].

This observation was confirmed in an independent study of 122 Han Chinese sibling pair families [149]. The authors believed that the (GT)n polymorphism in the NR2A promoter played a significant role in the etiology of schizophrenia. However, whether a trans factor interacts with these repeats is yet to be investigated. In another case-control study, a T-G variant within a proximal GC box of the NR2B promoter was correlated with schizophrenia [53]. In response to NGF treatment in PC12 cells, the T allele showed a 30-fold increase in promoter activity in comparison to the G allele. In a study of an Italian population, a G-C change of the first G in the GGGG sequence of a putative NFκB site in the 5′ UTR of the NR1 gene was correlated with human schizophrenia although its impact on promoter activity was not tested [150]. Polymorphisms of other NR subunits in schizophrenic patients have not yet been investigated.

Reduced expression of the NR genes was found in the brains of patients with other neurodegenerative disorders as well. Alzheimer’s disease patients showed reduced expression of the NR1 and NR2B gene in the hippocampus [151]. Parkinson’s patients exhibited reduced NR1 expression in the striatum and in the superficial layers of the prefrontal cortex [152]. Whether polymorphisms of the regulatory regions also play a role in this altered NR gene expression is an interesting question that remains to be addressed.

5.7.3. Alcoholism

Accumulating data indicate that glutamate neurotransmission may be damaged by ethanol inhibition. Expression of NR2B in the brain was found to be upregulated by ethanol treatment [153]. DNA methylation studies revealed that its promoter is demethylated in cultured neurons and in living animal models following chronic (but not acute) ethanol treatment [154,155]. The demethylation of two CpG islands upstream of the NR2B promoter is well correlated with the increase in NR2B mRNA. However, the functional effect of this demethylation on promoter activity has yet to be defined. Ethanol was also found to interfere with the inhibitory activities of RE1 clusters in the NR2B promoter [114] and enhance interactions of CREB and FosB with the AP1 site in the NR2B promoter [79], eventually upregulating the NR2B gene.

5.7.4. Hyperactivation by Agonist

Overstimulation of NRs by an abnormally high level of glutamate in nervous tissue has been hypothesized to mediate excitotoxicity in neurological disorders. However, hypoactivity of NR and reduced expression of NR genes was observed in a number of chronic neurological disorders and correlated with severity [140,141,143,151,152]. Whether the reductions are caused by glutamate overstimulation at the early stages of the disorders was investigated. Gascon et al. found that treatment of cultured cortical neurons with NMDA or glutamate resulted in a reduction of the NR1 protein and mRNA [156].

This effect was repeated on the NR1 promoter transfected into these cells. Mao et al. reported similar observations in cultured neurons and proposed that Sp1 protein and the proximal GC boxes were the mediators of this effect [157]. Treatment of cultured striatum neurons with quinolic acid, an endogenous NR agonist, produced the same effect [158]. This negative feedback in NR expression may be a defensive mechanism to avoid overactivation due to the persistent presence of agonist for neurons undergoing chronic pathological changes.

5.8. EPIGENETIC REGULATION

In a broad sense, the term epigenetics covers stable modifications of chromatin and DNA not involving DNA sequence change. DNA methylation, chromatin remodeling, and noncoding RNA are considered major epigenetic mechanisms [159].

5.8.1. DNA Methylation

Cytosine in a CpG dinucleotide is the major target of DNA methyltransferase in vertebrates [159,160]. Clusters of CpG sequences often reside in the promoters or their proximal regions to form so-called CpG islands [159,161]. CpG islands may become methylated and negatively regulate transcription. This pathway has been proposed as a critical epigenetic mechanism underlying development and other processes [159].

The CpG islands have been found within the promoter or proximal regions of the NR1, NR2A, NR2B, and NR2C genes [45,48,54,162]. The role of DNA methylation in promoter regulation has only been studied for the NR2B gene. The NR2B promoter was found to be hypermethylated in primary esophageal squamous cell carcinoma in which the NR2B is not expressed [162]. Demethylation by 5-aza-2′-deoxycytidine unmasked the promoter region and led to expression of NR2B transcripts as measured by reverse transcriptase polymerase chain reaction.

5.8.2. Chromatin Remodeling

Whether histones and other nuclear proteins regulate NR gene transcription via chromatin remodeling is largely unexplored. However, many trans factors found to directly interact with NR promoters such as REST and CREB are subject to regulation by chromatin remodeling [110,159]. Therefore, it can be hypothesized that this mechanism is also utilized to regulate NR transcription, but detailed direct evidence does not exist.

5.8.3. Noncoding RNA

Initial sequence analysis by TargetScan (http://www.targetscan.org) revealed that mRNAs of NR1, NR2A-D, and NR3A are included in the 30% of human mRNAs considered potential targets of miRNAs [163]. However, no studies have yet addressed directly the involvement of miRNAs in NR gene expression.

5.9. SUMMARY

Studies have revealed the functional activities of 5′ flanking sequences of the NR1, NR2A, 2B, and 2C genes using cultured cells and living animals. Interactions of several cis-acting regulatory elements with cognate trans factors and their impacts on promoter activity have been found to play important roles in NR gene expression under various conditions. Further studies should uncover additional pathways of differential NR expression. These studies will help explain the mechanisms of abnormal NR expression in human diseases and may also reveal potential pharmaceutical targets.

More than 1,000 transcription factors are dynamically expressed in the brain [164]. Thus it is likely that a network of these factors and interactions with a set of cis elements under the influence of epigenetic mechanisms ultimately determines NR gene expression under various conditions. A genome-wide approach may help address this question efficiently, particularly for investigating all functional cis elements involved in NR gene regulation. Those located in regions other than the 5′ flanking sequences, the 5′ UTR, and all functional regions of the NR2D and NR3A/B genes have not been systematically studied. A large-scale search for transcription regulatory regions may allow us to learn whether NR genes are involved in the complex transcription patterns uncovered by recent studies of the functional elements of the human genome (Figure 5.1) [19,44,165,166].

ACKNOWLEDGMENTS

The authors wish to thank Drs. Roland Dubner and Dean Dessem for critical reading of this manuscript and Dong Wei for drawing figures. GB was supported by NIH Grant NS38077 and by start-up funds from the University of Maryland Dental School.